Phagolysosome fusion is an important pathway in the degradation of internalized particles. Once a particle is internalized by phagocytosis it is directed toward the lysosomal compartment for degradation. Various studies have traced this sequence of events from binding and phagocytosis to eventual trafficking to lysosomes. In addition, the signaling machinery needed to perform many of these activities has been described. Recently, intracellular tyrosine-based activation motifs (ITAM) have taken center stage in the initiation and propagation of activation signals of phagocytic receptors.
ITAM motifs contribute to the ability of phagocytic receptors to efficiently internalize particles (Tuijnman et al, Blood 79:1651 (1992), Mitchell et al, Blood 84:1753 (1994)). ITAM motifs are composed of two YXXL motifs separated by a string of various amino acids. This motif forms a SH-2 binding domain for docking of signaling proteins such as Src and Syk, among others (Isakov Immunol. Res. 16:85 (1997), Isakov, J. Leuko. Biol. 61:6 (1997)). Specifically, upon ITAM phosphorylation, FcγRIIA has been shown to signal through Syk (Indik, et al, Blood 86:4389 (1995), Matsuda et al, Mol. Bio. Cell 7:1095 (1996)). In addition, mutation of either of the ITAM tyrosines abolishes the phagocytic activity of FcγRIIA (Mitchell et al, Blood 84:1753 (1994)). These YXXL sequences can also associate with adaptor proteins such as AP-1 and AP-2 in forming clathrin cups during phagocytosis.
Once a target is internalized, it can be sent to the lysosomal compartment for degradation. Di-leucine motifs in the cytoplasmic domain of various receptors are responsible for the trafficking of targets from phagosomes to lysosomes (Mayorga et al, J. Biol. Chem. 266:6511 (1991), Hunziker and Fumey, EMBO J. 13:2963 (1994), Letournier and Klausner, Cell 69:1143 (1992)). This motif is present in many receptors such as FcγRIIB, the LDL receptor, and the mannose 6-phosphate receptor (Matter et al, J. Cell Biol. 126:991 (1994), Johnson et al, J. Biol. Chem. 267:17110 (1992)). Mutation of either or both of the leucine residues in these receptors significantly reduces or abolishes lysosomal delivery, respectively.
FcγRIIA mediates phagocytosis through an ITAM motif and also mediates phagolysosomal fusion (Mitchell et al, Blood 84:1753 (1994)). However, there is no consensus di-leucine motif located in the cytoplasmic domain of FcγRIIA. Therefore, another sequence in the cytoplasmic domain of FcγRIIA must participate in lysosomal trafficking. The present invention relates to that sequence.